Methods and kits for reducing non-specific nucleic acid amplification

ABSTRACT

Methods and kits for efficient amplification of nucleic acids are provided. The methods comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having terminal mismatch primer-dimer structure. The methods also comprise in-vitro amplification of a nucleic acid template employing partially constrained primers having nucleotide analogues. The methods enhance efficiency of nucleic acid amplification reaction by reducing non-specific amplification reactions.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional of U.S. patent application, Ser. No. 12/337,746,which was filed on Dec. 18, 2008, and entitled METHODS AND KITS FORREDUCING NON-SPECIFIC NUCLEIC ACID AMPLIFICATION, which is herebyincorporated by reference in its entirety.

FIELD OF INVENTION

The invention generally relates to methods and kits for reducingnon-specific nucleic acid amplification for use in the field of nucleicacid amplification and detection. The methods described herein help toreduce primer interactions (e.g., primer-dimer structure formation)during nucleic acid amplification reactions, and thus reduce formationof false amplification products and background signals.

BACKGROUND

A variety of techniques are currently available for efficientamplification of nucleic acids even from a few molecules of a startingnucleic acid template. These include polymerase chain reaction (PCR),ligase chain reaction (LCR), self-sustained sequence replication (3SR),nucleic acid sequence based amplification (NASBA), strand displacementamplification (SDA), multiple displacement amplification (MDA), orrolling circle amplification (RCA). Many of these techniques involve anexponential amplification of the starting nucleic acid template, andgenerate a large number of amplified products in a short span of time.

Nucleic acid amplification techniques are often employed in nucleicacid-based assays used for analyte detection, sensing, forensic anddiagnostic applications, genome sequencing, whole-genome amplification,and the like. Such applications often require amplification techniqueshaving high specificity, sensitivity, accuracy, and robustness. However,most of the currently available techniques for nucleic acidamplification suffer from high background signals, which are generatedby non-specific amplification reactions yielding undesired/falseamplification products. These non-specific amplification reactionshinder effective utilization of many of these techniques in criticalnucleic acid-based assays. For example, if such an amplificationreaction were used for diagnostic applications, a false-positiveamplification (e.g., formation of amplification products even when thetemplate nucleic acid is absent) may likely result in a wrong diagnosis.Such non-specific, background amplification reactions become even moreproblematic where the target nucleic acid to be amplified is availableonly in limited quantities (e.g., whole-genome amplification from asingle DNA molecule).

Non-specific, background amplification reactions may be due toexogenous, non-target amplification (e.g., amplification of acontaminating nucleic acid), amplification of untargeted sequences, orprimer amplification (endogenous factors). A frequent source ofnon-specific amplification in a nucleic acid amplification reactionresults from various primer gymnastics. A primer may hybridize toregions of a nucleic acid (either in a target nucleic acid itself or ina contaminating nucleic acid) that share some homology with a targetedsequence of the target nucleic acid. If the 3′ end of a primer hassufficient homology to an untargeted region, the untargeted region mayget amplified. Non-specific amplification may also result from nucleicacid template-independent primer-primer interactions. Primers may formprimer-dimer structures by intra- or inter-strand primer annealing(intra molecular or inter molecular hybridizations), and may getamplified. The resultant spurious primer extension products may furtherget amplified, and may sometimes predominate, inhibit, or mask theamplification of the targeted sequence. In addition, duringamplification reaction, the amplification products may self-hybridize,allowing the nucleic acid polymerase to generate hybrid products orchimeric products.

Random primers (e.g., N₆, where N=A/T/G/C) are often used for nucleicacid amplification that demands amplification without significantsequence bias. They are useful for applications such as whole-genomeamplification, or for amplification of a target nucleic acid withunknown sequence. However, such random primers are also most susceptiblefor primer-dimer structure formation, and thus lead to higher levels ofnon-specific, endogenous background amplification. Hence, the use ofrandom primers in high efficiency nucleic acid amplification techniquesis often problematic. Constrained-randomized primers that cannotcross-hybridize via intra- or inter-molecular hybridization (e.g., R₆,where R=A/G) have been used for suppressing primer-dimer structureformation during nucleic acid amplification. However, suchconstrained-randomized primers impart considerable bias in nucleic acidamplification reaction in terms of sequence coverage. Such primers arealso of limited use for sequence-non-specific or sequence-non-biasednucleic acid amplification reactions (e.g., amplification ofwhole-genome, or amplification of a nucleic acid with unknown sequence).Thus, there exists a need for developing efficient nucleic acidamplification methods that have lower bias in terms of sequencecoverage, and have lower levels of non-specific, backgroundamplification. Development of primers that reduce primer-primerinteraction, and can support nucleic acid amplification without sequencebias is also needed.

BRIEF DESCRIPTION

One or more of the embodiments of the invention provide methods and kitsfor efficient amplification of nucleic acids. In some embodiments,methods for nucleic acid amplification employing primers having terminalmismatch primer-dimer structure are provided. In some embodiments, themethod comprises the steps of providing a nucleic acid template,contacting the nucleic acid template with a partially constrained primerhaving a terminal mismatch primer-dimer structure, and amplifying thenucleic acid template.

In some embodiments, methods for isothermal nucleic acid amplificationsusing a partially constrained primer comprising a nucleotide analogueare provided. In some embodiments, the method comprises the steps ofproviding a nucleic acid template, contacting the nucleic acid templatewith a partially constrained primer comprising a nucleotide analogue,and amplifying the nucleic acid template under isothermal conditions.

In some embodiments, methods for nucleic acid amplification areprovided, comprising the steps of providing a nucleic acid template;contacting the nucleic acid template with a nuclease-resistant,partially constrained primer to form a nucleic acid template-primercomplex; contacting the nucleic acid template-primer complex with aPhi29 polymerase and deoxyribonucleoside triphosphates; and amplifyingthe nucleic acid template. In some embodiments, the nuclease-resistant,partially constrained primer comprises a modified nucleotide, and alsoprovides a terminal mismatch primer-dimer structure.

In some embodiments, kits for nucleic acid amplification are provided.In some embodiments, the kit comprises a nucleic acid polymerase and apartially constrained primer having a terminal mismatch primer-dimerstructure.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a schematic illustration of some of the possible primer-dimerstructures that a partially constrained primer having terminal mismatchprimer-dimer structure may generate according to one embodiment of theinvention.

FIG. 2 shows a no template control (NTC) deoxyribonucleic acid (DNA)amplification reaction illustrating the reduction of non-specificnucleic acid amplification according to one embodiment of the invention.The figure illustrates the use of a partially constrained primercomprising LNA nucleotides that has a terminal mismatch primer-dimerstructure.

FIG. 3 shows higher amplification specificities of a DNA amplificationreaction when a partially constrained primer according to one embodimentof the invention is used in the DNA amplification reaction.

FIG. 4 shows single DNA molecule amplification using a partiallyconstrained primer having a terminal mismatch primer-dimer structureaccording to one embodiment of the invention.

FIG. 5 shows the stochastic nature of DNA amplification products when apartially constrained primer having a terminal mismatch primer-dimerstructure according to one embodiment of the invention is used in asingle DNA molecule amplification reaction.

FIG. 6 shows the amplification kinetics of various nuclease-resistant,partially constrained primers having terminal mismatch primer-dimerstructures. The partially constrained primers contain varying number ofphosphorothioate linkages.

FIG. 7 shows the restriction digestion pattern of the amplificationproducts when various nuclease-resistant partially constrained primershaving terminal mismatch primer-dimer structures were used for nucleicacid amplification. The partially constrained primers contain varyingnumber of phosphorothioate linkages.

FIG. 8 shows amplification efficiencies of various primers that comprisenucleotide analogues in a DNA amplification reaction according to someembodiments of the invention.

FIG. 9 shows DNA amplification efficiencies of a partially constrainedpentamer primer or partially constrained hexamer primers with respect tovarying target DNA template concentration according to some embodimentsof the invention.

DETAILED DESCRIPTION

Nucleic acid-based assays involving single molecule DNA amplification orwhole-genome amplification demand highly efficient nucleic acidamplification methods that have high yield, high fidelity and havelittle bias in terms of sequence coverage. A variety of methods that arecurrently available for use include, but are not limited to, polymerasechain reaction (PCR), ligase chain reaction (LCR), self-sustainedsequence replication (3SR), nucleic acid sequence based amplification(NASBA), strand displacement amplification (SDA), and rolling circleamplification (RCA). Isothermal nucleic acid amplification reactionssuch as rolling circle amplification (RCA), or multiple displacementamplification (MDA) employing random primers are more adaptable thantemperature-dependent nucleic acid amplification reaction (e.g., PCR)for such applications. However, these methods often yield a dominantbackground signal due to undesired non-specific nucleic acidamplification reactions, especially when the concentration of targetnucleic acid template is lower (e.g., below 1 ng).

One or more embodiments of the present invention are directed at methodsand kits for efficient amplification of nucleic acids. In someembodiments, the methods describe in-vitro amplification of a nucleicacid template that employ partially constrained primers having terminalmismatch primer-dimer structure. In some embodiments, the methodsdisclose in-vitro amplification of a nucleic acid template employingpartially constrained primers having nucleotide analogues. The methodsenhance the efficiency and sensitivity of a nucleic acid amplificationreaction by reducing non-specific amplification kinetics.

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms, which are used in the following description and theappended claims. Throughout the specification, exemplification ofspecific terms should be considered as non-limiting examples.

The singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. In some instances, theapproximating language may correspond to the precision of an instrumentfor measuring the value. Similarly, “free” may be used in combinationwith a term, and may include an insubstantial number, or trace amountswhile still being considered free of the modified term. Where necessary,ranges have been supplied, and those ranges are inclusive of allsub-ranges there between.

As used herein, the term “nucleoside” refers to a glycosylamine compoundwherein a nucleic acid base (nucleobase) is linked to a sugar moiety.The nucleic acid base may be a natural nucleobase or amodified/synthetic nucleobase. The nucleic acid base includes, but notlimited to, a purine base (e.g., adenine or guanine), a pyrimidine(e.g., cytosine, uracil, or thymine), or a deazapurine base. The nucleicacid base may be linked to the 1′ position, or at an equivalent positionof a pentose (e.g., a ribose or a deoxyribose) sugar moiety. The sugarmoiety includes, but is not limited to, a natural sugar, a sugarsubstitute (e.g., a carbocyclic or an acyclic moiety), a substitutedsugar, or a modified sugar (e.g., bicyclic furanose unit as in LNAnucleotide). The nucleoside may contain a 2′-hydroxyl, 2′-deoxy, or2′,3′-dideoxy forms of the sugar moiety.

As used herein the terms “nucleotide” or “nucleotide base” refer to anucleoside phosphate. It includes, but is not limited to, a naturalnucleotide, a synthetic nucleotide, a modified nucleotide, or asurrogate replacement moiety (e.g., inosine). The nucleoside phosphatemay be a nucleoside monophosphate, a nucleoside diphosphate or anucleoside triphosphate. The sugar moiety in the nucleoside phosphatemay be a pentose sugar, such as ribose, and the phosphate esterificationsite may correspond to the hydroxyl group attached to the C-5 positionof the pentose sugar of the nucleoside. A nucleotide may be, but is notlimited to, a deoxyribonucleoside triphosphate (dNTP) or aribonucleoside triphosphate (NTP). The nucleotides may be representedusing alphabetical letters (letter designation), as described inTable 1. For example, A denotes adenosine (i.e., a nucleotide containingthe nucleobase, adenine), C denotes cytosine, G denotes guanosine, and Tdenotes thymidine. W denotes either A or T/U, and S denotes either G orC. N represents a random nucleotide (i.e., N may be any of A, C, G, orT/U). A plus (+) sign preceding a letter designation denotes that thenucleotide designated by the letter is a LNA nucleotide. For example, +Arepresents an adenosine LNA nucleotide, and +N represents a lockedrandom nucleotide (a random LNA nucleotide). A star (*) sign preceding aletter designation denotes that the nucleotide designated by the letteris a phosphorothioate modified nucleotide. For example, *N represents aphosphorothioate modified random nucleotide.

TABLE 1 Letter designations of various nucleotides. Symbol LetterNucleotide represented by the symbol Letter G G A A T T C C U U R G or AY T/U or C M A or C K G or T/U S G or C W A or T/U H A or C or T/U B Gor T/U or C V G or C or A D G or A or T/U N G or A or T/U or C

As used herein, the term “nucleotide analogue” refers to compounds thatare structurally similar (analogues) to naturally occurring nucleotides.The nucleotide analogue may have an altered phosphoate backbone, sugarmoiety, nucleobase, or combinations thereof. Generally, nucleotideanalogues with altered nucleobases confer, among other things, differentbase pairing and base stacking proprieties. Nucleotide analogues havingaltered phosphate-sugar backbone (PNA, LNA) often modify, among otherthings, the chain properties such as secondary structure formation.

As used herein, the term “ LNA (Locked Nucleic Acid) nucleotide” refersto a nucleotide analogue, wherein the sugar moiety of the nucleotidecontains a bicyclic furanose unit locked in a ribonucleic acid(RNA)-mimicking sugar conformation. The structural change from adeoxyribonucleotide (or a ribonucleotide) to the LNA nucleotide islimited from a chemical perspective, namely the introduction of anadditional linkage between carbon atoms at 2′ position and 4′ position(e.g., 2′-C, 4′-C-oxymethylene linkage; see, for example, Singh, S. K.,et. al., Chem. Comm , 4, 455-456, 1998, or Koshkin, A. A., et. al.,Tetrahedron, 54, 3607-3630, 1998.)). The 2′ and 4′ position of thefuranose unit in the LNA nucleotide may be linked by an O-methylene(e.g., oxy-LNA: 2′-O, 4′-C-methylene-β-D-ribofuranosyl nucleotide), aS-methylene (thio-LNA), or a NH-mehtylene moiety (amino-LNA), and thelike. Such linkages restrict the conformational freedom of the furanosering. LNA oligonucleotides display enhanced hybridization affinitytoward complementary single-stranded RNA, and complementary single- ordouble-stranded DNA. The LNA oligonucleotides may induce A-type(RNA-like) duplex conformations.

As used herein, the term “oligonucleotide” refers to oligomers ofnucleotides or derivatives thereof. The term “nucleic acid” as usedherein refers to polymers of nucleotides or derivatives thereof. Theterm “sequence” as used herein refers to a nucleotide sequence of anoligonucleotide or a nucleic acid. Throughout the specification,whenever an oligonucleotide/nucleic acid is represented by a sequence ofletters, the nucleotides are in 5′→3′ order from left to right. Forexample, an oligonucleotide represented by a letter sequence(W)_(x)(N)_(y)(S)_(z), wherein x=2, y=3 and z=1, represents anoligonucleotide sequence WWNNNS, wherein W is the 5′ terminal nucleotideand S is the 3′ terminal nucleotide. The oligonucleotides/nucleic acidsmay be a DNA, a RNA, or their analogues (e.g., phosphorothioateanalogue). The oligonucleotides or nucleic acids may also includemodified bases, and/or backbones (e.g., modified phosphate linkage ormodified sugar moiety). Non-limiting examples of synthetic backbonesthat confer stability and/or other advantages to the nucleic acids mayinclude phosphorothioate linkages, peptide nucleic acid, locked nucleicacid, xylose nucleic acid, or analogues thereof.

As used herein, the term “terminal nucleotide” refers to a nucleotidethat is located at a terminal position of an oligonucleotide sequence.The terminal nucleotide that is located at a 3′ terminal position isreferred as a 3′ terminal nucleotide, and the terminal nucleotide thatis located at a 5′ terminal position is referred as a 5′ terminalnucleotide. The nucleotide adjacent to the terminal nucleotide refers toa nucleotide that is located at a penultimate position from the terminalposition.

As used herein, the term “primer”, or “primer sequence” refers to ashort linear oligonucleotide that hybridizes to a target nucleic acidsequence (e.g., a DNA template to be amplified) to prime a nucleic acidsynthesis reaction. The primer may be a RNA oligonucleotide, a DNAoligonucleotide, or a chimeric sequence. The primer may contain natural,synthetic, or modified nucleotides. Both the upper and lower limits ofthe length of the primer are empirically determined The lower limit onprimer length is the minimum length that is required to form a stableduplex upon hybridization with the target nucleic acid under nucleicacid amplification reaction conditions. Very short primers (usually lessthan 3 nucleotides long) do not form thermodynamically stable duplexeswith target nucleic acid under such hybridization conditions. The upperlimit is often determined by the possibility of having a duplexformation in a region other than the pre-determined nucleic acidsequence in the target nucleic acid. Generally, suitable primer lengthsare in the range of about 3 nucleotides long to about 40 nucleotideslong.

As used herein, the term “random primer” or “complete random primer”refers to a mixture of primer sequences, generated by randomizing anucleotide at any given location in an oligonucleotide sequence in sucha way that the given location may consist of any of the possiblenucleotides or their analogues (complete randomization). Thus the randomprimer is a random mixture of oligonucleotide sequences, consisting ofevery possible combination of nucleotides within the sequence. Forexample, a hexamer random primer may be represented by a sequence NNNNNNor (N)₆. A hexamer random DNA primer consists of every possible hexamercombinations of 4 DNA nucleotides, A, C, G and T, resulting in a randommixture comprising 4⁶ (4,096) unique hexamer DNA oligonucleotidesequences. Random primers may be effectively used to prime a nucleicacid synthesis reaction when the target nucleic acid's sequence isunknown.

As described herein, “partially constrained primer” refers to a mixtureof primer sequences, generated by completely randomizing some of thenucleotides of an oligonucleotide sequence (i.e., the nucleotide may beany of A, T/U, C, G, or their analogues) while restricting the completerandomization of some other nucleotides (i.e., the randomization ofnucleotides at certain locations are to a lesser extent than thepossible combinations A, T/U, C, G, or their analogues). For example, apartially constrained DNA hexamer primer represented by WNNNNN,represents a mixture of primer sequences wherein the 5′ terminalnucleotide of all the sequences in the mixture is either A or T. Here,the 5′ terminal nucleotide is constrained to two possible combinations(A or T) in contrast to the maximum four possible combinations (A, T, Gor C) of a completely random DNA primer (NNNNNN). Suitable primerlengths of a partially constrained primer may be in the range of about 3nucleotides long to about 15 nucleotides long.

As described herein, the term “partially constrained primer having aterminal mismatch primer-dimer structure” refers to a partiallyconstrained primer sequence, wherein when two individual primersequences in the partially constrained primer hybridize each otherinter-molecularly, with an internal homology of three or morenucleotides, to form a primer-dimer structure having no recessed ends,or a primer-dimer structure having a single-nucleotide base 3′ recessedends, or a primer-dimer structure having a two-nucleotide base 3′recessed ends, there exists a nucleotide mismatch (i.e., nucleotides donot base-pair) at both the 3′ terminal nucleotides in the primer-dimerstructure. For example, a partially constrained pentamer primerrepresented by WNNNS provides a terminal mismatch at both the 3′terminal nucleotides when it is inter-molecularly hybridized to form aprimer-dimer structure having no recessed ends. In the primer-dimerstructure, there exist an internal homology of three nucleotides (i.e.,the three random nucleotides in WNNNS may base-pair with each other whenthe primer-dimer structure having no recessed ends is formed byinter-molecular hybridization). However, this primer example does notprovide a terminal mismatch when it is inter-molecularly hybridized toform a primer-dimer structure with single-nucleotide base 3′ recessedends (see FIG. 1; rectangular boxes in the figure illustrates internalhomology of the primer-dimer structure). Similarly, a partiallyconstrained hexamer primer represented by WWNNNS provides a terminalmismatch at both the 3′ terminal nucleotides when it isinter-molecularly hybridized to form a primer-dimer structure having norecessed ends. Moreover, this primer example provides a terminalmismatch at both the 3′ terminal nucleotides even when it isinter-molecularly hybridized to form a primer-dimer structure having asingle-nucleotide base 3′ recessed ends. A partially constrainedheptamer primer represented by WWWNNNS provides a terminal mismatch atboth the 3′ terminal nucleotides when it is inter-molecularly hybridizedto form a primer-dimer structure having no recessed ends. Further, thisprimer example provides a terminal mismatch at both the 3′ terminalnucleotides when it is inter-molecularly hybridized to form aprimer-dimer structure having a single-nucleotide base 3′ recessed ends,or to form a primer-dimer structure having a two-nucleotide base 3′recessed ends.

As used herein, the term “plasmid” refers to an extra-chromosomalnucleic acid that is separate from a chromosomal nucleic acid. A plasmidDNA may be capable of replicating independently of the chromosomalnucleic acid (chromosomal DNA) in a cell. Plasmid DNA is often circularand double-stranded.

As used herein, the terms “amplification”, “nucleic acid amplification”,or “amplifying” refer to the production of multiple copies of a nucleicacid template, or the production of multiple nucleic acid sequencecopies that are complementary to the nucleic acid template.

As used herein, the term “target nucleic acid” refers to a nucleic acidthat is desired to be amplified in a nucleic acid amplificationreaction. For example, the target nucleic acid comprises a nucleic acidtemplate.

As used herein, the term “DNA polymerase” refers to an enzyme thatsynthesizes a DNA strand de novo using a nucleic acid strand as atemplate. DNA polymerase uses an existing DNA or RNA as the template forDNA synthesis and catalyzes the polymerization of deoxyribonucleotidesalongside the template strand, which it reads. The newly synthesized DNAstrand is complementary to the template strand. DNA polymerase can addfree nucleotides only to the 3′-hydroxyl end of the newly formingstrand. It synthesizes oligonucleotides via transfer of a nucleosidemonophosphate from a deoxyribonucleoside triphosphate (dNTP) to the3′-hydroxyl group of a growing oligonucleotide chain. This results inelongation of the new strand in a 5′→3′ direction. Since DNA polymerasecan only add a nucleotide onto a pre-existing 3′-OH group, to begin aDNA synthesis reaction, the DNA polymerase needs a primer to which itcan add the first nucleotide. Suitable primers comprise oligonucleotidesof RNA or DNA. The DNA polymerases may be a naturally occurring DNApolymerases or a variant of natural enzyme having the above-mentionedactivity. For example, it may include a DNA polymerase having a stranddisplacement activity, a DNA polymerase lacking 5′→3′ exonucleaseactivity, a DNA polymerase having a reverse transcriptase activity, or aDNA polymerase having an endonuclease activity.

As used herein the term “proofreading DNA polymerase” refers to any DNApolymerase that is capable of correcting its errors while performing DNAsynthesis. A proofreading DNA polymerase possesses a 3′→5′ exonucleaseactivity apart from its polymerase activity, and this exonucleaseactivity is referred as proofreading activity. Proofreading activity ofsuch polymerases correct mistakes in the newly synthesized DNA. DuringDNA synthesis, when an incorrect base pair is recognized, theproofreading DNA polymerase reverses its direction by one base pair ofDNA. The 3′→5′ exonuclease activity (proofreading activity) of theenzyme allows the incorrect nucleotide base pair to be excised.Following the nucleotide base excision, the polymerase re-inserts thecorrect nucleotide base, and continues the DNA synthesis. When freedNTPs are present in the solution or reaction mixture suitable for DNAsynthesis, the primary activity of the proofreading DNA polymerase isDNA synthesis. However, when dNTPs are not available for the DNAsynthesis reaction, the primary activity of the proofreading DNApolymerase may be its 3′→5′ exonuclease activity. Some of theproofreading DNA polymerases may require the presence of a divalentcation for their proofreading activity as well as for their polymeraseactivity. Suitable divalent cations that can switch on the proofreadingactivity of the proofreading polymerases include, but are not limitedto, magnesium or manganese.

As used herein, “ a strand displacing nucleic acid polymerase” refers toa nucleic acid polymerase that has a strand displacement activity apartfrom its nucleic acid synthesis activity. That is, a strand displacingnucleic acid polymerase can continue nucleic acid synthesis on the basisof the sequence of a nucleic acid template strand (i.e., reading thetemplate strand) while displacing a complementary strand that had beenannealed to the template strand.

As used herein, the term “complementary”, when used to describe a firstnucleic acid/oligonucleotide sequence in relation to a second nucleicacid/oligonucleotide sequence, refers to the ability of a polynucleotideor oligonucleotide comprising the first nucleic acid/oligonucleotidesequence to hybridize (e.g., to form a duplex structure) under certainhybridization conditions with an oligonucleotide or polynucleotidecomprising the second nucleic acid/oligonucleotide sequence.Hybridization occurs by base pairing of nucleotides (complementarynucleotides). Base pairing of the nucleotides may occur via Watson-Crickbase pairing, non-Watson-Crick base pairing, or base pairing formed bynon-natural/modified nucleotides.

As used herein the term “high stringent hybridization conditions” referto conditions that impart a higher stringency to an oligonucleotidehybridization event than the stringency provided by conditions that aregenerally used for nucleic acid amplification reactions. For example, ahigh stringent hybridization condition may be effected in a nucleic acidamplification reaction by increasing the reaction temperature or bydecreasing the salt concentration. Nucleic acid amplification reactionsare often carried out at about 75 mM salt concentration. In contrast, ifa nucleic acid amplification reaction is performed at about 15 mM saltconcentration, it may offer a high stringent hybridization condition.High stringent hybridization condition may be provided in an in-vitroisothermal nucleic acid amplification reaction by increasing thetemperature from about 30° C., which is often used. For example, theisothermal nucleic acid amplification reaction may be performed at about35° C. to about 45° C. to provide a high stringent hybridizationcondition.

As used herein, the term “rolling circle amplification (RCA)” refers toa nucleic acid amplification reaction that amplifies a circular nucleicacid template (e.g., single stranded DNA circles) via a rolling circlemechanism. Rolling circle amplification reaction may be initiated by thehybridization of a primer to a circular, often single-stranded, nucleicacid template. The nucleic acid polymerase then extends the primer thatis hybridized to the circular nucleic acid template by continuouslyprogressing around the circular nucleic acid template to replicate thesequence of the nucleic acid template over and over again (rollingcircle mechanism). The rolling circle amplification typically producesconcatamers comprising tandem repeat units of the circular nucleic acidtemplate sequence. The rolling circle amplification may be a linear RCA(LRCA), exhibiting linear amplification kinetics (e.g., RCA using asingle specific primer), or may be an exponential RCA (ERCA) exhibitingexponential amplification kinetics. Rolling circle amplification mayalso be performed using multiple primers (multiply primed rolling circleamplification or MPRCA) leading to hyper-branched concatamers. Forexample, in a double-primed RCA, one primer may be complementary, as inthe linear RCA, to the circular nucleic acid template, whereas the othermay be complementary to the tandem repeat unit nucleic acid sequences ofthe RCA product. Consequently, the double-primed RCA may proceed as achain reaction with exponential (geometric) amplification kineticsfeaturing a ramifying cascade of multiple-hybridization,primer-extension, and strand-displacement events involving both theprimers. This often generates a discrete set of concatemeric,double-stranded nucleic acid amplification products. The rolling circleamplification may be performed in-vitro under isothermal conditionsusing a suitable nucleic acid polymerase such as Phi29 DNA polymerase.

As used herein, multiple displacement amplification (MDA) refers to anucleic acid amplification method, wherein the amplification involvesthe steps of annealing a primer to a denatured nucleic acid followed bya strand displacement nucleic acid synthesis. As nucleic acid issynthesized by strand displacement, a gradually increasing number ofpriming events occur, forming a network of hyper-branched nucleic acidstructures. MDA is highly useful for whole-genome amplification forgenerating high-molecular weight DNA with limited sequence bias from asmall amount of genomic DNA sample. Strand displacing nucleic acidpolymerases such as Phi29 DNA polymerase or large fragment of the BstDNA polymerase may be used in multiple displacement amplification. MDAis often performed under isothermal reaction conditions, and randomprimers are used in the reaction for achieving amplification withlimited sequence bias.

As used herein the term “reaction mixture” refers to the combination ofreagents or reagent solutions, which are used to carry out a chemicalanalysis or a biological assay. In some embodiments, the reactionmixture comprises all necessary components to carry out a nucleic acid(DNA) synthesis/amplification reaction.

As used herein, the terms “reagent solution” or “solution suitable forperforming a DNA synthesis reaction” refer to any or all solutions,which are typically used to perform an amplification reaction or DNAsynthesis. They include, but are not limited to, solutions used inisothermal DNA amplification methods, solutions used in PCRamplification reactions, or the like. The solution suitable for DNAsynthesis reaction may comprise buffer, salts, and/or nucleotides. Itmay further comprise primers and/or a DNA template to be amplified.

One or more embodiments are directed to methods and kits for improvednucleic acid amplification reactions that are less prone to generatingfalse amplification products. These amplification methods are morereliable than currently available amplification techniques and so aremore suitable for applications such as amplification of rare sequences(i.e., where target nucleic acids are available in lower amount; e.g.,detection of rare mutant sequences within a population of wild-typesequences), or whole genome amplification reactions. The amplificationmethods use specially designed partially constrained primers to reducebackground amplification reactions. Suitable length of the partiallyconstrained primer may be in the range of about 3 nucleotides to about10 nucleotides long. In some embodiments, the partially constrainedprimer consists of about 5 nucleotides to about 7 nucleotides.

The partially constrained primers reduce the formation of stableprimer-dimer structures under conditions that are commonly used fornucleic acid amplification reactions. The reduction of stableprimer-dimer structures that may be extended during nucleic acidamplification reactions, in turn reduces the false amplificationreactions. The reduced levels of stable primer-dimer structures in thereaction mixture is achieved by designing the partially constrainedprimers in such a way that even if the primer-dimer structure is formedin the reaction mixture, it will be formed with a terminal mismatch. Ina primer-dimer structure having terminal mismatch, the 3′ terminalnucleotides are not base paired, and so is not amenable for primerextension reaction during nucleic acid amplification.

In some embodiments, the partially constrained primer comprises, atsuitable locations, nucleic acid analogues that have highercomplementary specificity than that of natural nucleotides (e.g., LNAnucleotides). The location of nucleotide analogues in the partiallyconstrained primer is chosen in such a way that it inhibits theformation of stable primer-dimer structures under nucleic acidamplification reaction conditions. Moreover, the nucleotide analoguesare positioned such that even if the partially constrained primerhybridizes inter-molecularly (to form a primer-dimer structure), thereoccurs a terminal mismatch even at high stringent hybridizationconditions. Hence, even if primer-dimer structures are formed duringnucleic acid amplification reaction, the formed primer-dimer structureswith terminal mismatch will not be extended during amplificationreaction due to the unpaired 3′ terminal nucleotides.

When the partially constrained primer comprising LNA nucleotide is usedfor nucleic acid amplification reaction, the amplification reaction maybe performed at more stringent hybridization conditions. Theamplification reaction may be performed at higher temperatures (e.g.,above 30° C. for an isothermal nucleic acid amplification), the upperlimit being the temperature at which the DNA polymerase used in thereaction may become non-functional. It may also be performed at a lowersalt concentration (e.g., about 10 μM to about 25 μM salt concentration)than what is normally used (e.g., about 75 μM salt concentration). Dueto higher complementary specificity, the hybridization of the partiallyconstrained primer comprising LNA nucleotides to the target nucleic acidwill not be substantially affected by high stringent hybridizationconditions. Hence, the amplification of the desired target nucleic acidamplification will also not substantially affected. The amenability ofusing stringent hybridization conditions further reduces the probabilityof formation of stable primer-dimer structures, and thus reducesnon-specific nucleic acid amplifications.

In some embodiments, improved methods and kits for isothermal nucleicacid amplification are provided. The methods and kits reducenon-specific nucleic acid amplification reactions by reducing primergymnastics. Non-limiting examples of suitable isothermal nucleic acidamplification reactions comprise rolling circle amplification (RCA) ormultiple displacement amplification (MDA). The methods may be used inthe amplification of circular nucleic acid templates or linear nucleicacid templates. The methods may be effectively used even when the amountof the nucleic acid template to be amplified is minimal. The methods maybe useful in whole-genome amplification or in single nucleic acidamplification reactions.

In one embodiment, a method for nucleic acid amplification is providedthat employs a partially constrained primer, which is designed to have aterminal mismatch primer-dimer structure. The method comprises providinga nucleic acid template, contacting the nucleic acid template with apartially constrained primer having a terminal mismatch primer-dimerstructure, and amplifying the nucleic acid template. Suitable length ofthe partially constrained primer may be in the range of about 3nucleotides to about 10 nucleotides long. In some embodiments, thepartially constrained primer may be a tetramer, pentamer, hexamer or aheptamer. A combination of partially constrained primers having varyingprimer lengths may also be used.

The method may further include the steps of addition of a nucleic acidpolymerase and deoxyribonucleoside triphosphates before theamplification step. The nucleic acid template may be amplified using anyof a variety of nucleic acid amplification methods. The amplification ofthe nucleic acid template may be performed using thermal cyclingmethods, such as polymerase chain reaction (PCR), or it may beperformed, at least in part, under isothermal conditions. In someembodiments, the nucleic acid template is amplified using isothermalnucleic acid amplification methods.

The nucleic acid polymerase that is used for amplification may be aproofreading or a non-proofreading nucleic acid polymerase. In someembodiments, the nucleic acid polymerase used is a strand displacingnucleic acid polymerase. The nucleic acid polymerase may be atermophilic or a mesophilic nucleic acid polymerase. Examples of DNApolymerases that are suitable for use include, but are not limited to,Phi29 DNA polymerase, hi-fidelity fusion DNA polymerase (e.g.,Pyrococcus-like enzyme with a processivity-enhancing domain, New EnglandBiolabs, MA), Pfu DNA polymerase from Pyrococcus furiosus (Strategene,Lajolla, Calif.), Bst DNA polymerase from Bacillus stearothermophilus(New England Biolabs, MA), Sequenase™ variant of T7 DNA polymerase,exo(−) Vent® DNA polymerase (New England Biolabs, MA), Klenow fragmentfrom DNA polymerase I of E. coli, T7 DNA polymerase, T4 DNA polymerase,DNA polymerase from Pyrococcus species GB-D (New England Biolabs, MA),or DNA polymerase from Thermococcus litoralis (New England Biolabs, MA).

In some embodiments, the methods may employ a highly processive,strand-displacing polymerase to amplify the nucleic acid template underconditions for high fidelity base incorporation. A high fidelity DNApolymerase refers to a DNA polymerase that, under suitable conditions,has an error incorporation rate equal to or lower than those associatedwith commonly used thermostable PCR polymerases such as Vent DNApolymerase or T7 DNA polymerase (from about 1.5×10⁻⁵ to about 5.7×10⁻⁵).Additional enzymes may be included in the amplification reaction mixtureto minimize mis-incorporation events. For example, protein mediatederror correction enzymes, such as, MutS, may be added to improve thepolymerase fidelity either during or following the polymerase reaction.

The nucleic acid template may be a linear template, nicked template or acircular template. It may be a natural or synthetic nucleic acid. It maycomprise a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). Insome embodiments, the nucleic acid template may be a DNA template. TheDNA template may be a cDNA or a genomic DNA. The circular nucleic acidtemplate may be a synthetic template (e.g., a linear or nicked DNAcircularized by enzymatic/chemical reactions), or it may be a plasmidDNA. In some embodiments, the method may amplify a circular DNA templateby rolling circle amplification. In some other embodiments, a linear DNAtemplate may be amplified using multiple displacement nucleic acidamplification.

In some embodiments, a Phi29 DNA polymerase or Phi29-like polymerase maybe used for amplifying a DNA template. In some embodiments, acombination of a Phi29 DNA polymerase and a Taq DNA polymerase may beused for the circular DNA amplification.

The partially constrained primers may be generated by completelyrandomizing (i.e., the nucleotide base may be any of A, T/U, C, G ortheir analogues) some nucleotides of an oligonucleotide sequence, whilerestricting the complete randomization of some other nucleotides (i.e.,the randomization of nucleotide bases at certain locations are to alesser extent than the four possible combinations A, T/U, C or G). Therandomization of the nucleotides is restricted in such a way that if thepartially constrained primer dimerizes, it forms a primer-dimerstructure that has a terminal mismatch. Thus, when two individual primersequences in the partially constrained primer hybridize to each otherinter-molecularly with an internal homology of three or more nucleotidebases, to form a primer-dimer structure having no recessed ends, or aprimer-dimer structure having a single-nucleotide base 3′ recessed ends,or a primer-dimer structure having a two-nucleotide base 3′ recessedends, there exists a nucleotide mismatch at both the 3′ terminalnucleotide bases in the primer-dimer structure (i.e., the 3′ terminalnucleotides in the primer dimer structure are unpaired.). Thisconstrained randomization at certain selected nucleotides reduce theprobability of the formation of stable primer-dimer structures in thereaction mixture during nucleic acid amplification. Moreover, even ifthe primer-dimer structures are formed during the nucleic acidamplification reaction, the terminal mismatch at both the 3′ terminalnucleotides in the primer-dimer structures hinders any primer extensionreaction by the nucleic acid polymerase from those 3′ terminalnucleotides.

In some embodiments, randomization of two nucleotides in the partiallyconstrained primer is restricted. In some embodiments, the randomizationof more than two nucleotides (e.g., three, four, or five nucleotides) inthe partially constrained primer is restricted. The extent ofrandomization may be empirically determined based on amplificationreaction requirements and conditions.

In some embodiments, the partially constrained primer is designed torestrict the randomization of terminal nucleotides. For example, thepartially constrained primer may be designed to consist of a sequence,W(N)_(y)S, wherein y=2, 3, 4 or 5. Here, the 5′ terminal nucleotide ofthe primer sequence is restricted to W (i.e., either A or T), and the 3′terminal nucleotide is restricted to S (i.e., either G or C). Since Wcannot base pair with S, there will be a terminal mismatch at both the3′ terminal nucleotides if the primer-dimer structure without anyrecessed ends is formed by inter-molecular hybridization. However, ifthe primers are inter-molecularly hybridized with at leastone-nucleotide base recessed ends, there will not be any terminalmismatch since S or W may base pair with N (i.e., S may base pair withN, when N is G or C; and W may base pair with N, when N is A or T).Non-limiting examples of primers, that have restricted randomizationonly at the terminal nucleotides include, W(N)_(y)S, S(N)_(y)W,D(N)_(y)G, G(N)_(y)D, C(N)_(y)A, or A(N)_(y)C. The integer value of ymay be in the range 2 to 13. In some embodiments, the value of y may be2, 3, 4, or 5. In one embodiment, the partially constrained primerconsists of a hexamer primer, the sequence of which may be WNNNNS,SNNNNW, DNNNNG, GNNNND, CNNNNA, or ANNNNC. In another embodiment, thepartially constrained primer consists of a pentamer primer, the sequenceof which may be WNNNS, SNNNW, DNNNG, GNNND, CNNNA, or ANNNC.

In some embodiments, the randomization of both of the terminalnucleotides of the partially constrained primer may be restricted alongwith a restricted randomization of the nucleotide that is adjacent tothe 5′ terminal nucleotide. For example, the partially constrainedprimer may be designed to consist of a sequence WW(N)_(y)S, wherein y=2,3, 4 or 5. Since W cannot base pair with S, there will be a terminalmismatch at both the 3′ terminal nucleotides if the primer-dimerstructure is formed (via inter-molecular hybridization) without anyrecessed ends. For some partially constrained primers, primer-dimerstructure may have a terminal mismatch even when primer-dimers areformed with recessed 3′ ends. For example, if a random primer of thesequence WWNNNS makes a primer-dimer structure even with asingle-nucleotide 3′ recessed ends, there will exist a nucleotidemismatch at both the 3′ terminal nucleotides. This will considerablyreduce the probability of stable primer-dimer structures in nucleic acidamplification reactions. Even if such primer-dimer structures are formedduring the amplification reaction, nucleic acid synthesis is notfeasible from any of the 3′ terminal nucleotides. Hence, thenon-specific nucleic acid amplification effected by primer-dimerstructure formation may be decreased considerably, which in turn mayresult in lower background amplification. The primers that haverestricted randomization at both terminal nucleotides and also at thenucleotide adjacent to the 5′ terminal nucleotide include, but are notlimited to, WW(N)_(y)S, SS(N)_(y)W, DD(N)_(y)G, GG(N)_(y)D, CC(N)_(y)A,or AA(N)_(y)C. The integer value of y may be in the range 2 to 12. Insome example embodiments, the value of y may be 2, 3, or 4. For example,the partially constrained primer may consists of a hexamer primer, thesequence of which may be WWNNNS, SSNNNW, DDNNNG, GGNNND, CCNNNA, orAANNNC. In some embodiments, the partially constrained primer mayconsists of a pentamer primer, the sequence of which may be WWNNS,SSNNW, DDNNG, GGNND, CCNNA, or AANNC.

In some embodiments, the partially constrained primer may be anuclease-resistant primer. For example, the partially constrained primermay be resistant to an exonuclease (e.g., a 3→′5′ exonuclease). In someembodiments, the partially constrained primer includes modification atthe sugar-phosphate backbone that makes the primer resistant to theexonuclease digestion. For example, the partially constrained primer maypossess one, two, three, or four phosphorothioate linkages betweennucleotides that are located toward the 3′ end of the primer sequence.In some embodiments, the partially constrained primer contains onephosphorothioate linkage that makes the primer resistant to degradationby an exonuclease. Non-limiting examples include WWNN*S, SSNN*W, DDNN*G,GGNN*D, CCNN*A, AANN*C, WWNNN*S, SSNNN*W, DDNNN*G, GGNNN*D, CCNNN*A, orAANNN*C. In some embodiments, the partially constrained primer containsmore than one phosphorothioate linkages in the sugar-phosphate backbone.Examples include partially constrained primer sequences such as, but arenot limited to, WWN*N*S, SSN*N*W, DDN*N*G, GGN*N*D, CCN*N*A, AAN*N*C,WWNN*N*S, SSNN*N*W, DDNN*N*G, GGNN*N*D, CCNN*N*A, or AANN*N*C. Themodification of the sugar-phosphate backbone in the sequence may be at a3′-terminal position, or it may be located at a position other than the3′-terminal position. When the modification is located at positionsother than the 3′-terminal end of a partially constrained primersequence, the 3′-terminal nucleotide may be removed by the 3′→5′exonuclease activity of an exonuclease. In some embodiments, multiplepartially constrained primers may be used for nucleic acidamplification. The multiple partially constrained primers may be chosenfrom partially constrained primers sensitive to exonuclease activity, orpartially constrained primers resistant to exonuclease activity. In someembodiments, a mixture of partially constrained primers sensitive toexonuclease activity and resistant to exonuclease activity may be usedfor amplification reaction.

In some embodiments, the partially constrained primer may comprise anucleotide analogue at a suitable position. In some embodiments, anucleotide analogue that has higher complementary specificity than thatof a natural nucleotide may be used. Non-limiting examples of suitablenucleic acid analogues that may be incorporated in the partiallyconstrained primer include peptide nucleic acids (PNA), 2′-fluoroN3-P5′-phosphoramidates, 1′,5′-anhydrohexitol nucleic acids (HNA), orlocked nucleic acid (LNA) nucleotides. The location of nucleotideanalogues in the partially constrained primer is chosen in such a waythat if the partially constrained primer hybridizes inter-molecularly(to form a primer-dimer structure), there occurs a terminal mismatcheven at stringent hybridization conditions (e.g., at a temperature thatis about 5° C. to about 10° C. higher than a typical nucleic acidamplification reaction condition). Moreover, the nucleotide analoguesare positioned such that the formation of stable primer-dimer structuresunder nucleic acid amplification reaction conditions are substantiallyinhibited. Due to higher complementary specificity of the nucleotideanalogues, a nucleic acid amplification reaction using partiallyconstrained primers comprising nucleotide analogues may be performed atmore stringent conditions (e.g. performing the reaction at highertemperatures or lower salt concentration). The partially constrainedprimer having nucleotide analogues has higher complementary specificityto the target (i.e., The T_(m) of the target nucleic acid-primer complexmay be higher when the partially constrained primer comprises thenucleotide analogue). Since such primer hybridizes to the target nucleicacid even at higher temperatures/lower salt concentration, the desiredtarget nucleic acid amplification is not substantially affected understringent hybridization conditions. Moreover, the amenability of usingstringent hybridization conditions for amplification reactions furtherreduces the probability of formation of stable primer-dimer structures.Furthermore, even if primer-dimer structures are formed during nucleicacid amplification reaction, they will be formed with a terminalmismatch and so will not be extended by the nucleic acid polymerase.

In some embodiments, the partially constrained primer comprises a LNAnucleotide at a suitable position. Suitable LNA nucleotides include, butare not limited to, an oxy-LNA (2′-O,4′-C-methylene-β-D-ribofuranosylnucleotide), a thio-LNA (2′-S,4′-C-methylene-β-D-ribofuranosylnucleotide), or an amino-LNA (2′-NH, 4′-C-methylene-β-D-ribofuranosylnucleotide) nucleotide. LNA nucleotide may be located toward the 5′ endof the partially constrained primer sequence. The partially constrainedprimer incorporating LNA nucleotide may form primer-dimer structureswith terminal mismatch. In some embodiments, the partially constrainedprimer comprises two LNA nucleotides. For example, a partiallyconstrained primer may have a LNA nucleotide at the 5′ terminalposition, and also at the position adjacent to the 5′ terminal position.In some other examples, the 5′ terminal nucleotide of the partiallyconstrained primer may be a natural nucleotide whereas the next twonucleotides adjacent to the 5′ terminal nucleotide may be LNAnucleotides. Non-limiting examples of partially constrained primerscomprising LNA nucleotides and having a primer-dimer structure with aterminal mismatch include, +W+WNNS, +S+SNNW, +D+DNNG, +G+GNND, +C+CNNA,+A+ANNC, +W+WNNNS, +S+WNNNW, +D+DNNNG, +G+GNNND, +C+CNNNA. +A+ANNNC,W+W+NNS, S+S+NNW, D+D+NNG, G+G+NND, C+C+NNA, A+A+NNC, W+W+NNNS,S+W+NNNW, D+D+NNNG, G+G+NNND, C+C+NNNA., or A+A+NNNC.

The partially constrained primer comprising a LNA nucleotide may be anexonuclease-resistant primer that is resistant to 3′→5′ exonucleaseactivity. They may possess a single or multiple phosphorothioatelinkages between nucleotides at the 3′ end of the primer sequence. Nonlimiting examples, include +W+WNN*S, +S+SNN*W, +D+DNN*G, +G+GNN*D,+C+CNN*A, +A+ANN*C, +W+WNNN*S, +S+WNNN*W, +D+DNNN*G, +G+GNNN*D,+C+CNNN*A. +A+ANNN*C, W+W+NN*S, S+S+NN*W, D+D+NN*G, G+G+NN*D, C+C+NN*A,A+A+NN*C, W+W+NNN*S, S+W+NNN*W, D+D+NNN*G, G+G+NNN*D, C+C+NNN*A.,A+A+NNN*C, +W+WN*N*S, +S+SN*N*W, +D+DN*N*G, +G+GN*N*D, +C+CN*N*A,+A+AN*N*C, +W+WNN*N*S, +S+WNN*N*W, +D+DNN*N*G, +G+GNN*N*D, +C+CNN*N*A.+A+ANN*N*C, W+W+N*N*S, S+S+N*N*W, D+D+N*N*G, G+G+N*N*D, C+C+N*N*A,A+A+N*N*C, W+W+NN*N*S, S+W+NN*N*W, D+D+NN*N*G, G+G+NN*N*D, C+C+NN*N*A.,or A+A+NN*N*C.

In some embodiments, a method for isothermal nucleic acid amplificationis provided using a partially constrained primer that comprises anucleic acid analogue. The method comprises the steps of providing anucleic acid template; contacting the nucleic acid template with apartially constrained primer, wherein the partially constrained primercomprises a nucleotide analogue; and amplifying the nucleic acidtemplate under isothermal conditions.

The partially constrained primer may be a partially constrained primerhaving a terminal mismatch primer-dimer structure. In one embodiment,the partially constrained primer is designed such that the nucleotidesat 3′ terminal position and 5′ terminal position are non-complementaryto each other. The 3′ terminal nucleotide of the partially constrainedprimer may further be non-complementary to a nucleotide adjacent to the5′ terminal nucleotide. In some embodiments, the partially constrainedprimer consisting of a nucleotide sequence (W)_(x)(N)_(y)(S)_(z), may beemployed. In this sequence, x, y and z are integer values independent ofeach other. In some embodiments, the values of x, y, and z in(W)_(x)(N)_(y)(S)_(z) may be x=2 or 3; y=2, 3 or 4; and z=1 or 2respectively.

In some embodiments, the nucleotide analogue in the partiallyconstrained primer comprises a LNA nucleotide. Non-limiting examplesinclude +W+WNNS, W+W+NNS, +W+WNNNS, or W+W+NNNS. In some embodiments,the partially constrained primer may be an exonuclease-resistant primer.In one embodiment, the partially constrained primer comprises aphosphorothioate linkage between a 3′ terminal nucleotide and anucleotide that is adjacent to the 3′ terminal nucleotide. In some otherembodiments, multiple phosphorothioate linkages may be present in thesequence. Non-limiting examples include W+W+NN*S, +W+WNN*S, W+W+NNN*S,+W+WNNN*S, W+W+N*N*S, +W+WN*N*S, W+W+NN*N*S, or +W+WNN*N*S.

The nucleic acid template may be a single-stranded nucleic acid templateor it may be a double-stranded nucleic acid template. It may be acircular nucleic acid template, a nicked nucleic acid template, or alinear nucleic acid template. The nucleic acid template may comprise aDNA, an RNA or a DNA-RNA chimeric template. The nucleic acid templatemay be a synthetic nucleic acid or a natural nucleic acid. It may alsocomprise modified nucleotides. The nucleic acid DNA template may bederived from a genomic DNA, an RNA template (using reverse transcriptaseenzymes) or a cDNA. In one example embodiment, the nucleic acid templateis a circular DNA template.

Non-limiting examples of isothermal nucleic acid amplification methodsinclude ligase chain reaction (LCR), self-sustained sequence replication(SSR), nucleic acid sequence-based amplification (NASBA), loop-mediatedisothermal amplification (LAMP), amplification with Qb-replicase, or thelike. In some embodiments, the nucleic acid template is amplified usingstrand displacement amplification reaction (SDA). In some embodiments,the nucleic acid template is amplified using multiple displacementamplification (MDA). In one embodiment, the nucleic acid template isamplified using rolling circle amplification (RCA) method. Rollingcircle amplification that could be used may be a linear RCA (LRCA) or itmay be an exponential RCA (ERCA). In another embodiment, multiply primedrolling circle amplification (MPRCA) is employed for amplifying thenucleic acid template.

The nucleic acid polymerase that may be employed in the isothermalnucleic acid amplification reaction may be a prokaryotic, fungal, viral,bacteriophage, plant, or eukaryotic nucleic acid polymerase. Suitablenucleic acid polymerases may also comprise holoenzymes, functionalportions of the holoenzymes, chimeric polymerase, or any modifiedpolymerase that can effectuate the synthesis of a nucleic acid molecule.Non-limiting examples of suitable DNA polymerases that may be usedinclude bacteriophage Phi29 DNA polymerase, Phi29-like polymerases(e.g., Phage M2 DNA polymerase, Phage B103 DNA polymerase, or Phage GA-1DNA polymerase), phage Phi-PRD1 polymerase, Vent DNA® polymerase (NewEngland Biolabs, MA), Deep Vent® DNA polymerase (New England Biolabs,MA), KlenTaq® DNA polymerase, DNA polymerase I, DNA polymerase Imodified with T7 DNA polymerase sequences, Klenow fragment of DNApolymerase I, DNA polymerase III, DNA polymerase III holoenzymes, T5 DNApolymerase, T4 DNA polymerase holoenzymes, T7 DNA polymerase,genetically engineered T7 DNA polymerase having reduced or insignificant3′→5′ exonuclease activity (e.g., Sequenase™ DNA polymerase), DNApolymerase form Thermoanaerobacter thermohydrosulfuricus (Tts DNApolymerase), or fragment thereof, modified Tts DNA polymerase, Bstpolymerase, rBST DNA polymerase, N29 DNA polymerase, or TopoTaq DNApolymerase.

In some embodiments, wherein the DNA template is a circular DNAtemplate, the circular DNA template may be amplified using a rollingcircle amplification method. Rolling circle amplification that may besuitable to use with the present invention includes a linear RCA (LRCA)or an exponential RCA (ERCA). In some example embodiments, multiplyprimed rolling circle amplification (MPRCA) is employed for amplifyingthe circular DNA template. In some embodiments, a ligation rollingcircle amplification is employed for amplifying the circular DNAtemplate. The rolling circle amplification of the circular DNA templatemay yield a concatamer comprising tandem repeat units of DNA templatesequence.

The DNA polymerase that may be used to amplify the circular DNA templatemay be, but is not limited to, a proofreading DNA polymerase or anon-proofreading DNA polymerase. In some embodiments, the proofreadingDNA polymerase comprises a thermally stable DNA polymerase. ProofreadingDNA polymerase may be a thermophilic DNA polymerase or a mesophilic DNApolymerase. In some embodiments, a combination of a proofreading DNApolymerase and a non-proofreading DNA polymerase may be used forefficient amplification of the DNA template. Any suitable proofreadingDNA polymerase may be used. Examples of proofreading polymerases thatare suitable for use include, but are not limited to, Phi29 DNApolymerase, hi-fidelity fusion DNA polymerase (e.g., Pyrococcus-likeenzyme with a processivity-enhancing domain from New England Biolabs,MA), Pfu DNA polymerase from Pyrococcus furiosus (Strategene, Lajolla,Calif.), Klenow fragment from DNA polymerase I of E. coli, T7 DNApolymerase, T4 DNA polymerase, DNA polymerase from Pyrococcus speciesGB-D (New England Biolabs, MA) and DNA polymerase from Thermococcuslitoralis (New England Biolabs, MA). Suitable examples ofnon-proofreading DNA polymerase that could be used include, but are notlimited to Taq DNA polymerase, Tts DNA polymerase, large fragment of BstDNA polymerase, exo (−) DNA Polymerase gene from Pyrococcus species GB-D(New England Biolabs, MA), or exo (−) DNA Polymerase from Thermococcuslitoralis (New England Biolabs, MA).

In some embodiments, a nucleic acid amplification is provided, whereinthe method comprises the steps of providing a nucleic acid template;contacting the nucleic acid template with a nuclease-resistant,partially constrained primer to form a nucleic acid template-primercomplex; contacting the nucleic acid template-primer complex with aPhi29 polymerase and deoxyribonucleoside triphosphates; and amplifyingthe nucleic acid template. The nuclease-resistant, partially constrainedprimer comprises a modified nucleotide and has a terminal mismatchprimer-dimer structure. The nucleic acid template may be amplified byrolling circle nucleic acid amplification, or by multiple displacementnucleic acid amplification.

In some embodiments, the nucleic acid template is amplified to generatean amplified nucleic acid, in a solution suitable for performing anucleic acid amplification reaction. The amplification reaction oftenemploys reagents such as a primer, a nucleic acid polymerase, and freenucleotides (e.g., dNTPs). The nucleic acid polymerase that is employedin the amplification reaction may be a proofreading nucleic acidpolymerase. In some embodiments, each of the reagents used in thenucleic acid amplification reaction may be pre-treated to remove anycontaminating nucleic acid sequences. In some embodiments, pre-treatmentof the reagents includes incubating the reagents in presence ofUltra-Violet radiation. In some embodiments, the reagents arede-contaminated by incubating the reagents in the presence of a nucleaseand its co-factor (e.g., a metal ion). Suitable nucleases include, butare not limited to, exonucleases such as exonuclease I or exonucleaseIII. Proofreading DNA polymerases that may be used in a DNAamplification reaction may be de-contaminated by incubating with adivalent metal ion (e.g., magnesium or manganese). The free nucleotidesemployed in nucleic acid template amplification may include naturalnucleotides (e.g., dATP, dGTP, dCTP, or dTTP) or their modifiedanalogues. Other components such as buffers, salts and the like may alsobe added to allow the nucleic acid amplification to occur efficiently.

In some embodiments, kits for nucleic acid amplification are provided.The kits contain reagents, packaged together, that are required topractice the presently described methods of nucleic acid amplification.In one embodiment, the kit comprises a nucleic acid polymerase and apartially constrained primer having a terminal mismatch primer-dimerstructure. The nucleic acid polymerase and the partially constrainedprimer may be packaged in a single vessel or they may be packaged inseparate vessels.

In one embodiment, the kit comprises a Phi29 DNA polymerase and apartially constrained primer having a terminal mismatch primer-dimerstructure, packaged together. The partially constrained primer in thekit may comprise a nucleotide analogue, such as a LNA nucleotide. Insome embodiments, the partially constrained primer is a DNA-LNA chimeraprimer. The partially constrained primer in the kit may be anuclease-resistant primer, for example, an exonuclease-resistant primer.These exonuclease-resistant primers in the kit may contain one or morephosphorothioate linkages between the nucleotides. In one embodiment,the kit comprises a partially constrained primer, the nucleotidesequence of which consists of W+W+NN*S, or W+W+NNN*S, and a Phi29 DNApolymerase.

The kit may further comprise reagents or reagent solutions required forperforming a nucleic acid amplification reaction. It may further includean instruction manual detailing the specific components included in thekit, or the methods for using them in nucleic acid amplificationreactions, or both.

EXAMPLES

Unless specified otherwise, ingredients described in the examples arecommercially available from common chemical suppliers. Exonuclease I,Exonuclease III, 10X NE buffer, and EcoR1 enzyme are commerciallyavailable from New England Biolabs, MA. SYBR Green 1, Pico Green, and 1Kb Plus DNA ladder are commercially available from Invitrogen.Single-Stranded Binding protein (SSB protein) is commercially availablefrom USB. Some abbreviations used in the examples section are expandedas follows: “mg”: milligrams; “ng”: nanograms; “pg”: picograms; “fg”:femtograms; “ag”: attograms; “zg”: zeptograms; “mL”: milliliters;“mg/mL”: milligrams per milliliter; “mM”: millimolar; “μM”: micromolar;“pM”: picomolar; “mmol”: millimoles; “pmol”: picomoles; “μL”:microliters; “min.”: minutes; “° C.”: degree Celsius; and “h.”: hours.

FIG. 1 is a schematic illustration of some possible primer-dimerstructures that a partially constrained primer having a terminalmismatch primer-dimer structure may form during nucleic acidamplification reactions. The figure shows the primer-dimer structures ofa partially constrained primer of the general formula(W)_(x)(N)_(y)(S)_(z), wherein x=1, 2 or 3, y=3 and z=1 (a pentamer, ahexamer or a heptamer) according to one embodiment of the invention. Therectangular boxes in the figure represent the nucleotides havinginternal homology in the primer-dimer structure.

FIG. 1 shows that when two individual primer sequences in a partiallyconstrained primer, (W)_(x)N₃S, wherein x=1, 2, or 3, consisting of nnucleotides (n is a variable number; e.g., for a hexamer primer, n=6)and having a terminal mismatch primer-dimer structure, hybridize eachother inter-molecularly, with an internal homology of three or morenucleotides, to form a hybridization complex, either with no 3′ recessedends, or with (n-5)-nucleotide base long 3′ recessed ends, there exist anucleotide mismatch at both the 3′ terminal nucleotides in thehybridization complex.

If a primer-dimer structure of a partially constrained pentamer primerhaving a sequence WNNNS is formed via inter-molecular hybridization,with no recessed ends, both the 3′ terminal nucleotides will not be ableto base pair (since W cannot base pair with S) in the primer-dimerstructure. If such a hybridization event occurs during a nucleic acidamplification reaction conditions, a terminal mismatch primer-dimerstructure is yielded. However, as shown in FIG. 1, if the primer WNNNSforms a primer-dimer structure with a single-nucleotide base 3′ recessedends, there may not be any terminal mismatch at both the 3′ terminalnucleotides (i.e., S may base pair with N).

Similarly, if a partially constrained hexamer primer (n=6), having anoligonucleotide sequence WWNNNS, forms a primer-dimer structure with norecessed ends, both the 3′ terminal nucleotides in the primer-dimerstructure will not be able to base pair (since W cannot base pair withS). If such a hybridization event occurs, it will yield a terminalmismatch primer-dimer structure. Moreover, even if the primer WWNNNSforms a primer-dimer structure with a single-nucleotide base 3′ recessedends, terminal mismatch will exist at both the 3′ terminal nucleotidesin the primer-dimer structure. However, as illustrated in FIG. 1, theprimer-dimer structures of WWNNNS having more than one-nucleotide base3′ recessed ends, will not have a terminal mismatch at the 3′ terminalnucleotides.

A primer-dimer structures of a partially constrained heptamer primerWWWNNNS, host terminally mismatched 3′ terminal nucleotides (unpaired 3′terminal nucleotides) in the primer-dimer structure if a primer-dimerstructure is formed with no recessed ends, or with single-nucleotidebase 3′ recessed ends, or with two-nucleotide base 3′ recessed ends(see. FIG. 1). However, the partially constrained heptamer primer,WWWNNNS will not have unpaired 3′ terminal nucleotides if theprimer-dimer structure formed by the inter-molecular hybridization has3′ recessed ends consisting of more than three nucleotides.

Example 1

The reagents and reagent solutions that were used for nucleic acidamplification reaction were de-contaminated in a nucleic acid-free hoodprior to their use to remove any contaminating nucleic acids. Thereagents such as Phi29 DNA polymerase, exonuclease I, exonuclease III,and SSB protein were stored in 50 mM Tris-HCl (pH 7.2), 200 mM NaCl, 10mM DTT, 1 mM EDTA, 0.01% (v/v) Tween-20, and 50% (v/v) glycerol. Theprimer-nucleotide solution (primer-nucleotide mix) comprising primer andnucleotides (dNTPs) was de-contaminated by incubating theprimer-nucleotide mix with a combination of exonuclease I, exonucleaseIII, and a single stranded DNA binding protein (SSB protein). The enzymemix comprising a DNA polymerase was de-contaminated by incubating withan exonuclease in presence of a divalent cation (e.g., Mg²⁺). Any targetnucleic acid amplification reaction was performed using thede-contaminated enzyme mix and the primer-nucleotide mix.

As shown in Table 2, the enzyme mix containing 200 ng of Phi29 DNApolymerase was incubated with 0.1 unit of exonuclease III in 50 mM HEPESbuffer (pH=8.0) containing 15 mM KCl, 20 mM MgCl₂, 0.01% (v/v) Tween-20,and 1 mM TCEP (Total volume was 5 μL). The incubation was performedeither at 30° C. for about 60 min, or at 4° C. for 12 h. The incubatedenzyme mix was then transferred to an ice-bath, and was used in DNAamplification reactions as such without any inactivation of theexonuclease III. This small amount of exonuclease III had no substantialeffect on the amplification reaction if the finished amplificationreaction was treated immediately upon completion to inactivate theexonuclease.

To de-contaminate the primer-nucleotide mix, it was incubated with acombination of exonuclease I, exonuclease III and SSB protein as shownin Table 1. The incubation was performed at 37° C. for about 60 min. in50 mM HEPES buffer (pH=8.0) containing 15 mM KCl, 20 mM MgCl₂, 0.01%(v/v) Tween-20 and 1 mM TCEP (Total reaction volume was 5 μL). E. ColiSSB protein was used in this example as a suitable single-strandedbinding protein. After decontamination of the primer-nucleotide mix, theexonucleases were thermally inactivated by incubating theprimer-nucleotide mix at 85° C. for about 15 min., followed byincubation at 95° C. for about 5 min to about 10 min

For nucleic acid amplification reaction, the primer-nucleotide mix andthe enzyme mix was mixed together along with template nucleic acid tocreate an amplification reaction, which was then incubated at about 30°C. The time at which the signal in each reaction reached a specificminimal fluorescent intensity was identified.

For reactions with rapid kinetics, the signal appears more quickly; andfor reactions with slower kinetics (such as those with lower levels ofinput template DNA), the signal appears more slowly. In these reactions,and specific for each reaction, there is a relationship between theamount of DNA template used and the time at which the signal appears.Additionally, the time required for signal to appear in reactions thatcontain no input template DNA is an indication of how efficiently thereaction can produce an artificial template by some de-novo process, andthen amplify the artificial template (non-specific amplification).Reaction modifications that retain rapid kinetics in the presence oftemplate DNA, yet slow kinetics of reactions that lack template DNA aredesirable.

TABLE 2 De-contamination of the enzyme mix and primer-nucleotide mixsolutions. Primer- DNA polymerase nucleotide mix (enzyme) mix (eachreaction) (each reaction) 2X Reaction buffer (reaction buffer 2.5 μL 2.5μL is 50 mM HEPES buffer (pH = 8.0), 15 mM KCl, 20 mM MgCl₂, 0.01%Tween-20 and 1 mM TCEP) Distilled water — 2.2 μL 10 mM dNTP mix 0.4 μL —1 mM primer 0.4 μL — Exonuclease I (20 unit/μL) 0.5 μL — Exonuclease III(10 unit/μL) 0.1 μL — Exonuclease III (1 unit/μL) — 0.1 μL SSB protein(100 ng/μL)   1 μL — 1:100 SYBR Green I 0.1 μL — Phi29 DNA polymerase (1mg/ml) — 0.2 μL Total reaction volume   5 μL   5 μL

Example 2

Non-specific amplification reaction during a nucleic acid amplificationreaction was estimated by performing a DNA amplification reactionwithout any added template DNA (No Template Control (NTC)amplification). The reactions employed either a completely randomhexamer primer (NNNN*N*N), or a partially constrained pentamer primer(W+W+N*N*S) that comprises LNA nucleotides, which has a terminalmismatch primer-dimer structure. Both these primers wereexonuclease-resistant primers, having phosphorothioate linkages betweenthe nucleotides toward the 3′ end of the sequence.

The amplification products from a DNA amplification reaction with noadded target DNA template (NTC) arise from non-specific amplificationreactions (false amplification or background amplification). Thenon-specific amplification may be due to amplification of contaminatingDNA molecules, or it may be due to primer gymnastics (e.g., formation ofprimer-dimer structures, and subsequent extension of primer-dimers). Toavoid any non-specific amplification reaction originating fromcontaminating DNA, all the reagents or reagent solutions (enzyme mix andprimer-nucleotide) that were used for the amplification reaction werede-contaminated to remove any contaminating DNA using the proceduredescribed in Example 1.

For estimating non-specific DNA amplification reactions, DNAamplification reaction was performed by incubating the de-contaminatedprimer-nucleotide mix and the de-contaminated enzyme mix at 30° C. forabout 400 min without any added DNA template. The amplification reactionmixture was composed of 40 μM primer (random hexamer, NNNN*N*N, orpartially constrained pentamer, W+W+N*N*S); 400 μM dNTPs (equal mixtureof each of dATP, dCTP, dGTP, dTTP); and 0.3 μM phi29 DNA polymerase (200ng per 10 μL, reaction). The incubation was performed in 50 mM HEPESbuffer (pH =8.0) containing 15 mM KCl, 20 mM MgCl₂, 0.01% (v/v)Tween-20, 1 mM TCEP, and 1:10,000 (v/v) SYBR Green I. Real-time data wascollected in Tecan fluorescent plate reader by monitoring the SYBR Greenfluorescence using an excitation wavelength of 485 nm and an emissionwavelength of 535 nm

The time at which the fluorescent signal in each reaction has reached aspecific minimal fluorescent intensity is graphically represented inFIG. 2. The time required for fluorescent signal to appear in reactionsthat contain no input template DNA is an indication of how efficientlythe reaction can produce an artificial template by some de-novo processand then amplify the artificial template (non-specific amplificationreaction).

FIG. 2 shows that the partially constrained pentamer primer comprisingLNA nucleotides (W+W+N*N*S) decreased the non-template amplificationkinetics. When the amplification reaction employed the random hexamerprimer, NNNN*N*N, DNA amplification products (non-specific amplificationproducts) were observed at about 200 min. In contrast, when partiallyconstrained pentamer primer W+W+N*N*S was used for the amplificationreaction, the non-specific amplification products appeared only afterabout 300 min. This illustrates that DNA amplification at zero targetDNA concentration (the false amplification products) was considerablyreduced when partially constrained primer having a terminal mismatchprimer-dimer structure was employed.

Since all the reagents were de-contaminated prior to DNA amplificationreaction, significant contribution toward non-specific amplificationoriginates from primer gymnastics under amplification reactionconditions. Hence, reduction of non-specific amplification productsindicates reduced primer gymnastics. Multiple factors may contribute tothe reduction of primer-primer interaction in a partially constrainedprimer having terminal mismatch primer-dimer structure. The reduction ofthe length of the primer from a hexamer to a pentamer reduces thepossibility of formation of stable primer-dimer structures under nucleicacid amplification reaction conditions. Moreover, the partiallyconstrained W+W+N*N*S, when hybridized inter-molecularly with norecessed ends or with a single nucleotide 3′ recessed ends, hosts aterminal mismatch primer dimer structure. Such primer-dimer structures,if formed, will have 3′ terminal nucleotides that are not base-paired.Hence, formation of such primer-dimer structures may be limited.Furthermore, incorporation of LNA nucleotides in the partiallyconstrained pentamer primer increases the binding specificity of theprimer to the complementary sequences. Higher specificity primerscomprising LNA nucleotides allow the amplification reaction to beperformed at lower salt concentration of about 15 mM to about 20 mM(high stringent hybridization conditions) as compared to the commonlyused salt concentration of about 75 mM. In this example, theamplification reactions were performed at a lower salt concentration ofabout 15 mM salt. The reduction in salt concentration results in higherstringency hybridization conditions so that the chances of formation ofstable primer-dimer structures under the amplification reactionconditions are further reduced. Moreover, since the partiallyconstrained primer has a terminal mismatch primer-dimer structure, evenif primer-dimer structures were formed, primer dimer extension reactionswould not have been possible due to mismatched 3′ terminal nucleotides.

Example 3

Higher specificities of DNA amplification with a partially constrainedprimer is illustrated by performing DNA amplification reactions atvarying concentrations of target DNA template. To avoid any non-specificamplification reaction from contaminating DNA, all reagents or reagentsolutions (enzyme mix and primer-nucleotide mix) that were used for theamplification reaction were de-contaminated prior to their use followingthe procedure described in Example 1. The de-contaminated enzyme mix andprimer-nucleotide mix were then combined to form a reaction mixture,which was comprised of 40 μM primer (either a random hexamer, NNNN*N*N,or a partially constrained pentamer primer, W+W+N*N*S), 400 μM dNTPs(equal mixture of each of dATP, dCTP, dGTP, dTTP), and 0.3 μM phi29 DNApolymerase (200 ng per 10 μL reaction) in 50 mM HEPES buffer (pH=8.0)containing 15 mM KCl, 20 mM MgCl2, 0.01% (v/v) Tween-20, 1 mM TCEP, and1:10,000 (v/v) SYBR Green I. The reaction mixture also contained 0.1unit exonuclease III, 1 unit of exonuclease III (heat-inactivated), 10units of exonuclease I (heat-inactivated) and 100 ng E. coli SSB protein(heat-inactivated) from the de-contamination procedure.

Combined enzyme mix and primer-nucleotide mix were placed in low-bindingPCR tubes on ice (12 tubes, each having 10 μL reaction mixture). Serialdilutions of a plasmid DNA were performed so as to have varying numbersof plasmid DNA molecules in each of the PCR tubes. Briefly, 1 μL of 1ng/μL pUC19 plasmid (about 3×10⁸ DNA circles) was added to the firsttube. The reaction mixture was mixed well, and 1 μL was transferred fromthe first tube to the second tube to achieve a 10-fold serial dilution(i.e., about 3×10⁷ DNA circles). This process was repeated from tube 2to tube 3 and continued till tube 11. In the 12^(th) tube, no templateDNA was added and thus served as no template control (NTC).

For DNA amplification, from each tube, 10 μL reaction mixture wastransferred to a 384-well, flat bottom plate (384 Black from BD FalconMicrotest) with a transparent cover. It was then incubated in SniPer(Tecan) at about 30° C. The parameters used were; Gain: Manual-40; Lagtime: 0 μs; Z-position: Manual-8700; Integration time: 40 μs; # Flash:10 ms; Time between move: 0 ms; cycles: 50, 00:10:00 each. Real-timedata was collected in Tecan fluorescent plate reader by monitoring theSYBR Green fluorescence using an excitation wavelength of 485 nm and anemission wavelength of 535 nm.

FIG. 3 shows the higher amplification efficiencies of the DNAamplification reaction when the partially constrained primer, W+W+N*N*Swas used in the DNA amplification reaction. The figure shows that theno-template amplification (NTC) was reduced considerably in the reactionemploying W+W+N*N*S in comparison with the reaction employing the randomprimer, NNNN*N*S. The reduction in no-template amplification (background, non-specific amplification) may be attributed to the reductionin the formation of stable primer-dimer structure when the partiallyconstrained primer, W+W+N*N*S having terminal mismatch primer-dimerstructure was used for DNA amplification. Moreover, even if theprimer-dimer structure were formed when using W+W+N*N*S (either with norecessed 3′ ends or with a single nucleotide length 3′ recessed ends),due to 3′ terminal nucleotide mismatch, no primer extension reactionswould have been possible during DNA amplification. This is in contrastthe usage of NNNN*N*S, wherein formation of primer-dimer structures andtheir extensions are possible during DNA amplification reactionconditions.

FIG. 3 also illustrates that the DNA amplification efficiencies of thepartially constrained primer, W+W+N*N*S, was comparable to that ofrandom primer, NNNN*N*S when the average number of target DNA circleswas above 1×10⁰ DNA circles. This illustrates that W+W+N*N*S isefficient in reducing non-specific amplification reactions (averagenumber of DNA circles below 1×10⁰) and so may be effectively used forsingle molecule DNA amplification.

Example 4

Efficiency of a partially constrained primer for use in single moleculeDNA amplification was demonstrated by amplification of a mixture ofplasmids, pUC19 and pGEM-3Z-f(−). By diluting the mixture of plasmids,one should eventually reach a concentration at which the reactionmixture shall have only one of the two plasmids. Thus, by seriallydiluting the plasmid mixture, the dilution (or concentration) at whichthe amplification of only a single plasmid of the plasmid mixture occurswas identified.

To avoid any non-specific amplification reaction from contaminating DNA,all reagents or reagent solutions (enzyme mix and primer-nucleotide)that were used for the amplification reaction were de-contaminated priorto their use following the procedure described in Example 1. Thede-contaminated enzyme mix and primer-nucleotide mix were then combinedto form a reaction mixture (total volume 10 μL), which was comprised of40 μM primer (either a random hexamer, NNNN*N*N or a partiallyconstrained pentamer, W+W+N*N*S), 400 μM dNTPs (equal mixture of each ofdATP, dCTP, dGTP, dTTP), and 0.3 μM phi29 DNA polymerase (200 ng per 10μL reaction) in 50 mM HEPES buffer (pH=8.0) containing 15 mM KCl, 20 mMMgCl2, 0.01% (v/v) Tween-20, 1 mM TCEP, and 1:10,000 (v/v) SYBR Green I.The reaction mixture also contained 0.1 unit exonuclease III, 1 unit ofexonuclease III (heat-inactivated), 10 units of exonuclease I(heat-inactivated) and 100 ng E. coli SSB protein (heat-inactivated)from the de-contamination procedure.

The reaction mixture (combined enzyme mix and primer-nucleotide mix) wasplaced in low-binding PCR tubes on ice (12 tubes, each having 10 μLreaction mixture). Serial dilutions of a mixture of plasmid DNAs (pUC19and pGEM-3Z-f(−)) were performed in the reaction mixture so as to havevarying numbers of plasmid DNA molecules in each of the PCR tubes.Briefly, 1 μL of 1 ng/μL pUC19 plasmid and pGEM-3Z-f(−) plasmid mixture(1 ng plasmid DNA is about 3×10 ⁸ DNA circles) was added to the firsttube. The reaction mixture was mixed well, and 1 μL from the first tubewas transferred to the second tube to achieve a 10-fold serial dilution.The second tube will then have about 3×10⁷ DNA circles. This process wasrepeated from tube 2 to tube 3, and continued till tube 11. Predictedaverage number of DNA molecules that each tube may contain due to serialdilution is illustrated in Table 3. In the 12^(th) tube, no template DNAwas added and thus served as no template control (NTC).

TABLE 3 Concentration and predicted number of DNA molecules upon 10-foldserial dilutions. Tube Concentration Predicted avarge number Number ofDNA of DNA molecules 1 1 ng 3 × 10⁸ 2 100 pg 3 × 10⁷ 3 10 pg 3 × 10⁶ 4 1pg 3 × 10⁵ 5 100 fg 3 × 10⁴ 6 10 fg 3 × 10³ 7 1 fg 3 × 10² 8 100 ag 3 ×10¹ 9 10 ag 3 × 10⁰ 10 1 ag 3 × 10⁻¹ 11 100 ag 3 × 10⁻² 12 0 0 (NoTemplate Control)

For DNA amplification, 10 μL reaction mixture from each tube wastransferred to a 384-well, flat bottom plate (384 Black from BD FalconMicrotest) having a transparent cover. It was then incubated in SniPer(Tecan) at about 30° C. The parameters used were; Gain: Manual-40; Lagtime: 0 μs; Z-position: Manual-8700; Integration time: 40 μs; # Flash:10 ms; Time between move: 0 ms; and cycles: 50, 00:10:00 each. Real-timedata was collected in Tecan fluorescent plate reader by monitoring theSYBR Green fluorescence using an excitation wavelength of 485 nm and anemission wavelength of 535 nm.

Following the amplification, the plate was spun down in a centrifuge,and the samples were transferred to 0.2 mL PCR strip tubes. Phi29 DNApolymerase in the reaction mixture was denatured by incubating the tubesat about 80° C. for about 30 min. The tubes were then cooled to roomtemperature by keeping them at room temperature for about 30 min.

The amplification products in each tube were restriction digested usingEcoR1 enzyme. Briefly, 2 μL of amplified product was mixed with 1 μL ofEcoR1 (10 Units/μL), 1 μL 10× NE buffer, and 6 μL distilled water (totalvolume of 10 μL). The mixture was incubated at 37° C. for 1 h. Thedigested products in each tube was mixed with 3 μL of loading dye (2 μL6× loading buffer+1 μL, 1:200 PicoGreen) and loaded in 0.8% agarose gel.1 Kb Plus DNA Ladder was used in the marker lane (9 μL TE buffer, 2 μL6× loading buffer, 1 μL 1:200 PicoGreen and 1 μL of 1 Kb Plus DNA Ladder(1 μg/μL) were mixed together and loaded 10 μL on the gel).

FIG. 4 shows the agarose gel of the EcoR1 restriction digest ofamplification products of mixtures of pUC19 (2686 base pairs) andpGEM-3Z-f(−) (3197 base pairs). By diluting the mixture of plasmids, onewill eventually reach a concentration at which the reaction mixture willhave only one of the two plasmids. It can be seen from FIG. 4 that whenpartially constrained primer, W+W+N*N*S was used for amplification,amplification products were obtained from reaction mixtures thatseemingly had only one of the two plasmids in it (Lane 11: amplificationfrom 1 ag of DNA). Lane 11 shows the amplification products from pUC19(circled) and does not show any amplification products that correspondto pGEM DNA. This indicates that at this dilution, the reaction mixturehad only pUC19 DNA and did not contain any pGEM DNA. In contrast, therandom hexamer primer, NNNN*N*S did not facilitate amplification fromsingle/few DNA molecules. Amplification reactions employing NNNN*N*S didnot have enough sensitivity to reach that level and specific productswere no longer made below the 1000 DNA molecule level. This indicatesthat the partially constrained primer, W+W+N*N*S having a terminalmismatch primer-dimer structure was more efficient in amplificationreactions having smaller amount of target template DNA.

FIG. 5 demonstrates the stochastic nature of single molecule DNAamplification when W+W+N*N*S was employed in the amplification reaction.As shown in FIG. 4, by serially diluting the plasmid mixture, it waspossible to find out a dilution at which only one of the plasmids in theplasmid mixture got amplified (e.g., DNA concentration of 1 ag).However, when multiple, independent DNA amplification reactions wereperformed at such dilutions, the amplification reactions were found tobe stochastic. Few of the amplification reactions using 1 ag of templateDNA circles amplified both the plasmids, some amplified onlypGEM-3Z-f(−), whereas some others amplified only pUC19. Someamplification reactions did not amplify any of the two plasmids, andshowed non-specific amplification reactions (background amplifications).

Example 5

Amplification efficiencies of a partially constrained primer thatconsist of only one phosphorothioate linkage between a 3′ terminalnucleotide and a nucleotide that is adjacent to the 3′ terminalnucleotide is compared with that of a partially constrained primerconsisting of multiple phosphorothioate linkages.

DNA amplification reactions at varying concentration of a mixture ofplasmids, pUC19 and pGEM-3Z-f(−) were performed using primers W+W+N*N*Sor W+W+NN*S. To avoid any non-specific amplification reaction fromcontaminating DNA, all reagents or reagent solutions (enzyme mix andprimer-nucleotide) that were used for the amplification reaction werede-contaminated prior to their use following the procedure described inExample 1. The procedure used for DNA amplification reaction andsubsequent restriction digestion were essentially the same as describedin Example 4. By diluting the mixture of plasmids, one should eventuallyreach a concentration at which the reaction mixture shall have only oneof the two plasmids.

When a reaction to form a phosphorothioate linkage between twonucleotides is performed, only about 50% the nucleotide linkages willactually be exonuclease-resistant. This is due to the fact that in aphosphorothioation reaction only 50% of the linkages are eventuallyphosphorothioated, making them exonuclease resistant. Hence, when apartially constrained primer is designed to contain two phosphorothioatebonds (e.g., W+W+N*N*S), during its chemical synthesis, some of theprimer sequences (in the partially constrained mixture of sequences) mayactually end up with having only one phosphorothioate linkage instead ofthe designed two (e.g., W+W+NN*S or W+W+N*NS). Hence, there exists apossibility that some of the sequences may have a terminal 3′nucleotide, having a linkage that is exonuclease sensitive (e.g.,W+W+N*NS). Under such situation, the de-contamination procedures asdescribed in Example 1, may remove the 3′ terminal nucleotide ofW+W+N*NS and may yield a partially constrained primer that hascompletely randomized 3′ terminal nucleotide (W+W+N*N). The tetramerproduct, W+W+N*N then support primer-dimer structures having no terminalmismatch and may support extension reactions from its primer-dimerstructures. However, a partially constrained primer having a sequenceW+W+NN*S, will not generate such unwanted sequences in the reactionmixture. During the chemical synthesis of W+W+NN*S, some sequences maybe generated having a sequence W+W+NNS. However, suchexonuclease-sensitive sequences will be completely digested during thede-contamination procedure. Hence, a partially constrained primerconsisting of a single phosphorothioate linkage does not generateunwanted sequences during de-contamination procedures, and so wasexpected to reduce non-specific amplification reaction considerably.

However, as shown in FIG. 6 and FIG. 7, non-specific amplificationreactions were not reduced when a partially constrained primer thatconsists of only one phosphorothioate linkage between a 3′ terminalnucleotide and a nucleotide that is adjacent to the 3′ terminalnucleotide (W+W+NN*S) was employed for DNA amplification reactions. Notemplate amplification (NTC) surfaced at about 175 min. when W+W+NN*Swas used. However, when W+W+N*N*S was used for DNA amplification, notemplate amplification was observed only after about 230 min. FIG. 7shows that non-specific amplification was more when W+W+NN*S wasemployed in a nucleic acid amplification with 1 ag template DNA.

Example 6

Nucleic acid amplification efficiencies of various exonuclease-resistantprimers (random primer or partially constrained primers) comprisingnucleotide analogues were estimated by comparing their DNA amplificationefficiencies with or without added template DNA. To avoid anynon-specific amplification reaction from contaminating DNA, all reagentsor reagent solutions (enzyme mix and primer-nucleotide) that were usedfor the amplification reaction were de-contaminated prior to their usefollowing the procedure described in Example 1.

DNA amplification reactions were performed following the proceduresprovided in previous examples. For estimating non-specific DNAamplification reactions, DNA amplification reactions were performedwithout adding any template DNA (no template control). For amplificationreactions containing a target DNA template, 0.5 pg of pUC19 plasmid DNAwas used as a suitable template.

The amplification reaction mixture was composed of 40 μM primer; 400 μMdNTPs (equal mixture of each of dATP, dCTP, dGTP, dTTP); and 0.3 μMphi29 DNA polymerase (200 ng per 10 μL reaction) in 50 mM HEPES buffer(pH=8.0) containing 15 mM KCl, 20 mM MgCl2, 0.01% (v/v) Tween-20, 1 mMTCEP, and 1:10,000 (v/v) SYBR Green I. The DNA amplification wasperformed by incubating the reaction mixture (with or without addedtemplate DNA) at about 30° C. for about 400 min. Real-time data wascollected in Tecan fluorescent plate reader by monitoring the SYBR Greenfluorescence using an excitation wavelength of 485 nm and an emissionwavelength of 535 nm.

The time at which the signal in each reaction reached a specific minimalfluorescence intensity is identified, and is shown in FIG. 8. Forreactions with rapid kinetics, the signal appears more quickly; and forreactions with slower kinetics the signal appears more slowly.Additionally, the time required for the signal to appear in reactionsthat contain no input template DNA is an indication of how efficientlythe reaction can produce an artificial template by some de-novo processand then amplify the artificial template (non-specific amplification).Reaction modifications that retain rapid kinetics in the presence oftemplate DNA, yet slow kinetics of reactions that lack template DNA, aredesirable.

FIG. 8 shows the amplification kinetics of DNA amplification reactionswith different primers. In amplification reactions employing a randomprimer, NNN*N*N the fluorescent signal from non-template controlappeared almost at the same time as that of amplification reactionhaving 0.5 pg of template DNA. This indicates that the reaction kineticsin the presence of 0.5 pg of template DNA is almost the same as that ofreaction lacking template DNA. When primers such as N+N+N+N*+N*+N, orWWN+N*+N*S, wherein LNA nucleotides were located toward 3′ ends, wereused for amplification reaction, the no template amplification kineticswere significantly slower. The fluorescent signals were visible onlyafter about 450 min. of the reaction. However, these primers alsoreduced the kinetics of the amplification reaction even with 0.5 pg oftemplate DNA (The fluorescent signals appeared only after about 150 min.of reaction). Primers wherein the LNA nucleotides were positioned towardthe 5′ end of the primer sequence (e.g., +W+WNN*S, +W+WN*N*S,V+W+NN*N*S, W+W+NN*N*S, or W+W+NNN*S), demonstrated significantly betteramplification efficiencies. These primers slowed down the kinetics ofthe no template reaction without significantly slowing down the kineticsof amplification reaction in the presence of template DNA.

Example 7

DNA amplification efficiencies of some partially constrained primershaving terminal mismatch primer-dimer structures are illustrated byperforming DNA amplification reactions at varying concentrations oftarget template DNA. To avoid any non-specific amplification reactionsas a result of contaminating DNA, all reagents or reagent solutions(enzyme mix and primer-nucleotide) that were used for the amplificationreaction were de-contaminated prior to their use following the proceduredescribed in Example 1.

Varying concentrations of pUC19 plasmid DNA (Og (NTC), 25 pg, 250 fg,2.5 fg, 25 ag, or 250 zg) were placed in low binding PCR tubes. The tubewithout added pUC19 DNA served as no template control (NTC). DNAamplification reactions were performed using each of the primersfollowing the amplification procedures described in previous examples.Briefly, each of the amplification reactions contained, apart from thetarget DNA, 40 μM partially constrained primer (W+W+NN*N*S, V+W+N*N*S orW+W+N*N*S), 400 μM dNTPs (equal mixture of each of dATP, dCTP, dGTP,dTTP), and 0.3 μM phi29 DNA polymerase (200 ng per 10 μL reaction). Theamplification reaction was performed in 50 mM HEPES buffer (pH=8.0)containing 15 mM KCl, 20 mM MgCl2, 0.01% (v/v) Tween-20, 1 mM TCEP, and1:10,000 (v/v) SYBR Green I, by incubating the reaction mixture at about30° C. Real-time data was collected in Tecan fluorescent plate reader bymonitoring the SYBR Green fluorescence using an excitation wavelength of485 nm and an emission wavelength of 535 nm.

Following the amplification, the amplification products were restrictiondigested using EcoR1. Briefly, 2 μL of amplified product was mixed with1 μL of EcoR1 (10 Units/μL), 1 μL 10× NE buffer, and 6 μL distilledwater (total volume of 10 μL). The mixture was incubated at 37° C. for 1h. The digested products in each tube was mixed with 3 μL of loading dye(2 μL 6× loading buffer+1 μL, 1:200 Pico Green) and loaded in 0.8%agarose gel. 1 Kb Plus DNA Ladder was used in the marker lane (9 μL TEbuffer, 2 μL 6× loading buffer, 1 μL 1:200 Pico Green and 1 μL of 1 KbPlus DNA Ladder (1 μg/μL) were mixed together and loaded 10 μL on thegel).

FIG. 9 shows the agarose gel of the EcoR1 restriction digest ofamplification products using various primer compositions. The pentamerprimer W+W+N*N*S was found to be marginally better than that of thehexamer primer W+W+NN*N*S.

The precise use, choice of reagents, choice of variables such asconcentration, volume, incubation time, incubation temperature, and thelike may depend in large part on the particular application for which itis intended. While only certain features of the invention have beenillustrated and described herein, it is to be understood that oneskilled in the art, given the benefit of this disclosure, will be ableto identify, select, optimize or modify suitable conditions for usingthe methods in accordance with the principles of the present invention,suitable for these and other types of applications.

The foregoing examples and embodiments are illustrative of some featuresof the invention rather than limiting on the invention described herein.They are selected embodiments or examples from a manifold of allpossible embodiments or examples. The invention may be embodied in otherspecific forms without departing from the spirit or essentialcharacteristics thereof. It is, therefore, to be understood that theappended claims are intended to cover all modifications and changes thatfall within the true spirit of the present invention.

1. A kit for nucleic acid amplification, comprising: a nucleic acidpolymerase; and a primer solution, wherein the primer solution consistsessentially of a partially constrained primer having a terminal mismatchprimer-dimer structure.
 2. The kit of claim 1, wherein the nucleic acidpolymerase comprises a strand displacing nucleic acid polymerase.
 3. Thekit of claim 1, wherein the nucleic acid polymerase is a Phi29 DNApolymerase.
 4. The kit of claim 1, wherein the partially constrainedprimer is a hexamer primer, a pentamer primer, a tetramer primer, or acombination thereof.
 5. The kit of claim 1, wherein the partiallyconstrained primer consists of a nucleotide sequence(W)_(x)(N)_(y)(S)_(z), wherein x, y and z are integer values independentof each other, and wherein value of x is 2 or 3, value of y is 2, 3 or4, and value of z is 1 or
 2. 6. The kit of claim 5, wherein thepartially constrained primer is a pentamer primer, the nucleotidesequence of which consists of WWNNS.
 7. The kit of claim 5, wherein thepartially constrained primer is a hexamer primer, the nucleotidesequence of which consists of WWNNNS.
 8. The kit of claim 1, wherein thepartially constrained primer comprises a nucleotide analogue.
 9. The kitof claim 1, wherein the partially constrained primer is a DNA-LNAchimera primer.
 10. A kit for isothermal nucleic acid amplification,comprising: a nucleic acid polymerase; and a primer solution, whereinthe primer solution consists essentially of a partially constrainedprimer mixture, and wherein the partially constrained primer mixturecomprises a nucleotide analogue.
 11. The kit of claim 10, wherein thenucleotide analogue is a LNA nucleotide.
 12. The kit of claim 10,wherein the partially constrained primer comprises a 3′ terminalnucleotide and a 5′ terminal nucleotide, which are non-complementary toeach other.
 13. The kit of claim 12, wherein the 3′ terminal nucleotideof the partially constrained primer is further non-complementary to anucleotide adjacent to the 5′ terminal nucleotide.
 14. The kit of claim10, wherein the partially constrained primer is a pentamer primer, thenucleotide sequence of which consists of +W+WNNS, or W+W+NNS.
 15. Thekit of claim 10, wherein the partially constrained primer is a hexamerprimer, the nucleotide sequence of which consists of +W+WNNNS, orW+W+NNNS.
 16. The kit of claim 10, wherein the partially constrainedprimer comprises a phosphorothioate linkage between a 3′ terminalnucleotide and a nucleotide that is adjacent to the 3′ terminalnucleotide.
 17. The kit of claim 16, wherein the partially constrainedprimer is a pentamer primer, the nucleotide sequence of which consistsof W+W+NN*S, or +W+WNN*S.
 18. The kit of claim 16, wherein the partiallyconstrained primer is a hexamer primer, the nucleotide sequence of whichconsists of W+W+NNN*S, +W+WNNN*S.
 19. The kit of claim 16, wherein thepartially constrained primer is a pentamer primer, the nucleotidesequence of which consists of W+W+N*N*S, or +W+WN*N*S.
 20. The kit ofclaim 16, wherein the partially constrained primer is a hexamer primer,the nucleotide sequence of which consists of W+W+NN*N*S, or +W+WNN*N*S.21. A kit for isothermal nucleic acid amplification, comprising: anucleic acid polymerase; and a primer solution, wherein the primersolution consists essentially of a nuclease-resistant, partiallyconstrained primer mixture, wherein the nuclease-resistant partiallyconstrained primer comprises a modified nucleotide, and wherein thenuclease-resistant, partially constrained primer has a terminal mismatchprimer-dimer structure.